Nanoliposomes containing three essential oils from the Artemisia genus as effective larvicides against Aedes aegypti and Anopheles stephensi

Aedes aegypti and Anopheles stephensi have challenged human health by transmitting several infectious disease agents, such as malaria, dengue fever, and yellow fever. Larvicides, especially in endemic regions, is an effective approach to the control of mosquito-borne diseases. In this study, the composition of three essential oil from the Artemisia L. family was analyzed by Gas Chromatography–Mass Spectrometry. Afterward, nanoliposomes containing essential oils of A. annua, A. dracunculus, and A. sieberi with particle sizes of 137 ± 5, 151 ± 6, and 92 ± 5 nm were prepared. Besides, their zeta potential values were obtained at 32 ± 0.5, 32 ± 0.6, and 43 ± 1.7 mV. ATR-FTIR analysis (Attenuated Total Reflection-Fourier Transform InfraRed) confirmed the successful loading of the essential oils. Moreover, The LC50 values of nanoliposomes against Ae. aegypti larvae were 34, 151, and 197 µg/mL. These values for An.stephensi were obtained as 23 and 90, and 140 µg/mL, respectively. The results revealed that nanoliposomes containing A. dracunculus exerted the highest potential larvicidal effect against Ae. aegypti and An. stephensi, which can be considered against other mosquitoes.

www.nature.com/scientificreports/ beetle (Tribolium casteneum) 22 . Likewise, the insecticidal effect of A. dracunculus L. EO on Aphis gossypii was reported 23 . Moreover, the toxic and repellent potential of A. sieberi against Dermanyssus gallinae (poultry red mite) was reported 24 . Nanoliposome are bilayered vesicles containing amphiphilic molecules similar to cell membranes; stability, durability, potency, and efficacy of the entrapped compounds in nanoliposomes are improved [25][26][27] . Besides, some reports on applying nanoliposomes containing EOs as larvicides have been reported. For instance, nanoliposomes containing Citrus aurantium EO with LC 50 value of 4.9 µg/ml against Culex quinquefasciatus 28 . Moreover, nanoliposomes containing carvacrol with an LC 50 value of 128 µg/mL against An. stephensi 29 . However, to the authors' best knowledge, no report was available on the use of nanoliposomes containing EO as larvicides against Ae. aegypti.
For the first time, nanoliposomes containing A. annua, A. dracunculus, and A. sieberi EOs were proposed in the current study. Moreover, their larvicidal properties against Ae. aegypti and An. stephensi was investigated.

Materials and methods
Materials. Tween 20,cholesterol, and egg lecithin were obtained from Merck Chemicals Co. (Germany). The Culicidae insectary at Hormozgan University Medical Sciences supplied An. stephensi and Ae. aegypti larvae; they were continually available for bioassay testing. Besides, the mosquito colonies were maintained at relative humidity (70 ± 5%), with photoperiod cycles of 12:12 (light:dark) at 27 ± 1 °C. Moreover, the polytetrafluoroethylene (PTFE)-based membrane method was used to blood-feed adult female mosquitoes 30 . Chemical compositions of the EOs. The chemical compound of the EOs was performed using an Agilent type 6890 GC-MS device equipped with a BPX5 silica capillary column (30 m × 0.25 μm, layer thickness of 0.25 μm) as described in our previous report 31 . To identify the EO's constituent compositions, 1 µL n-hexane was added in column chromatography. The temperature was scheduled; the oven temperature was set to 50 °C for 5 min. Then, the temperature was increased to 240 °C at a rate of 3 °C min −1 , in continue, the temperature was increased to 300 °C at a rate of 15 °C min −1 for 3 min. Finally, the transfer line temperature was adjusted to 250 °C by split 1 to 35. Helium was used as the carrier gas at a flow rate of 0.5 mL min −1 . The mass spectrometer (Agilent 5973 model) was scanned between 40 to 500 amu with an ionization voltage of 70 eV and ionization source temperature of 220 °C. The software used was Chemstation. Identification of the spectra was done with the help of their inhibition index and its comparison with the indices found in the source books and papers, using the mass spectra of standard compounds and the information available in the computer and virtual library 32 .
Preparation of nanoliposomes containing EOs. Nanoliposomes were prepared using ethanol injection method 28 . The mixture of lecithin (3% w/v), cholesterol (0.5% w/v), tween 20 (2% w/v), and each EO (2% w/v) was added in absolute ethanol and stirred overnight (2000 RPM) to oily phase prepared. After that, 1 mL of the oily phase was added to the 4 mL of distilled water. Finally, the mixture was mixed for 1 h at 2000 RPM and room temperature to stabilize the formed nanoliposomes. Meanwhile, free nanoliposomes were prepared using the above method but without EO.
Investigation of size and zeta potential of the nanoliposomes. Three prepared nanoliposomes and free nanoliposomes were poured into a quartz cell and transferred to the DLS machine to investigate particle size and particle size distribution (SPAN). The SPAN was calculated through the following equation: d90 − d10/d50. Also, the zeta potential of the samples was measured at room temperature 33 . Investigation loading of the EOs in nanoliposomes. The loading of EOs was confirmed by ATR-FTIR qualitative method. For this purpose, each EO, free liposome, and nanoliposome containing each EO was subjected to the FTIR machine, and spectra in the 400 to 4000 wavenumber cm −1 were recorded 34 . Mosquito rearing and larvicidal bioassays. The World Health Organization-recommended protocol was applied for the larvicidal bioassay tests 35 . In a 400 mL beaker containing 200 mL water, 25 larvae of An. stephensi or Ae. aegypti in the late third and early fourth instars were subjected to 12.5, 25, 50, 100, and 20 µg/mL of nanoliposome containing each EO. Besides, 1 mL of ethanol and free nanoliposomes were added to three bakers as control and negative control groups. Larval mortality was then noted after 24 h exposure. Three replicates for larvicidal bioassay were carried out, and larvicidal effects are presented as mean ± standard deviations. Besides, LC 50 values with upper and lower confidence limits were calculated using the CalcuSyn software (free version). The non-overlap between the samples' upper and lower confidence limits was considered a significant difference. Ethical approval. The ethics committee has approved this research at Fasa University of Medical Sciences, Iran, IR.FUMS.REC.1401.145. Besides, this study did not include human investigation, so consent to participate is not applicable.

Results
Identified compounds in the EOs. Identified Fig. 2A-D; obtained values were 32 ± 0.5, 32 ± 0.6, 43 ± 1.7, and 23 ± 1.2 mV. Furthermore, due to the high concentration, the sedimentation in all three nanoliposomes started after about 6 h. After overnight, two-phase suspensions were observed, with a clear supernatant and an agglomerate of nanoparticles below; no oily phases were observed at the top of the solutions. Agglomerate differs from aggregate; in the first one, the boundary between the nanoparticles is preserved and can be re-dispersed. In the next one, the nanoparticles become one and cannot be re-dispersed easily 36,37 . However, due to the presence of surfactant in the prepared nanoliposomes, these suspensions were re-dispersed with a simple shake, and their size did not change much from the initial state (data not shown). It is added that because the preparation site of nanoliposomes (Fasa University of Medical Sciences, Iran) and the larvicide testing site (Hormozgan University of Medical Sciences, Iran) are about 500 km away, the larvicide tests were conducted after 6 months of preparation. Therefore, it can be concluded that this re-dispersion did not affect nanoliposome efficacy. However, if the larvicidal test could have been performed immediately after preparation, the results would have been more accurate. However, in www.nature.com/scientificreports/ practical conditions, a larvicide usually is used for several months or years after manufacturing, so when the efficacy of these nanoliposomes was proper after six months, it can be concluded that they have good stability.
Successful loading of the EOs in the nanoliposomes. ATR-FTIR spectrum of A. annua EO (Fig. 3A) displayed the broadband at 3514 cm −1 attributed to OH and the characteristic peaks at 3084 and 3028 cm −1 can be related to C-H SP 2 hybrid of alkyne. Besides, the spectra at 2963 and 2928 cm −1 corresponded to -CH stretching vibration due to alkanes, the spectra at 2872 and 2725 cm −1 indicating CH stretching vibration in aldehyde structure, the spectrum at 1743 cm −1 , related to C=O, and the spectra at 1646, 1445 cm −1 can be related to C=C in aromatic compounds. The peak at 1445 cm −1 is attributed to the bending vibration of alcohol C-OH. The peaks at 1167, 1071, and 1004 cm −1 can be related to the stretching vibrations of C-O. The spectrum at 970 cm −1 is allocated to =C-H out-of-plane bending vibration from aromatics, and the characteristic band at 749 cm −1 is attributed to C-H vibration in benzene. ATR-FTIR spectrum of A. dracunculus EO (Fig. 3B) displayed the peaks at 3076 and 3032 cm −1 ; they are attributed to SP 2 hybrid of alkyne, the spectra at 2976, 2953, 2933, 2906, and 2834 cm −1 displayed -CH stretching vibration in SP 3 . The characteristic band at 1727 and 1638 cm −1 can be allocated to carbonyl groups. The band at 1509 cm −1 can be related to the C=C vibration in the aromatic ring and the characteristic band at 1243 cm −1 is attributed to C-O stretching vibration. Besides, the spectrum at 1035 cm −1 can be related to C-H bending absorption; also, the spectrum at 808 cm −1 is allocated to C-H vibration in benzene.
ATR-FTIR spectrum of A. sieberi EO (Fig. 3C) indicated the spectrum at 3467 cm −1 assigned to OH stretching due to phenolic compound in the EO. The characteristic peaks at 2958, 2924, and 2872 m −1 are ascribed to C-H stretching due to aliphatic compound, and the strong band at 1741 corresponded to (C=O), Carbonyl stretch representing aldehyde or ketones. The peak at 1454 cm −1 exhibited CH 2 bending, and the absorption at about 1367 cm −1 is allocated to CH 3 bending.
ATR-FTIR spectrum of free liposome (Fig. 3D) displayed the broad band between 3200 and 3600 cm −1 attributed to the presence of the hydroxyl group (OH), and the spectra at 2977, 2929, and 2900 cm −1 are attributed to C-C-H stretching. The absorption at 1645 cm −1 corresponded to the presence of the carbonyl group, and the spectrum at 1453 cm −1 indicates CH 2 bending. Besides, the absorption at around 1383 cm −1 can be related to CH 3 bending. The characteristic spectrum at 1085 cm −1 confirmed that the presence of P=O, the absorption at 1044 cm −1 could be related to C-O stretching, the characteristic absorption at 934 cm −1 corresponded to N(CH 3 ) 3 , and the spectrum at 877 cm −1 represented the P-O stretching due to presence of lecithin. www.nature.com/scientificreports/ It is evidenced from the blank and liposome containing EO for all absorption bands of interest that little difference was observed because the functional group of EO overlapped with the strong bands of the blank liposome.
ATR-FTIR spectrum of liposome containing A. annua EO (Fig. 3E) represented the broad and characteristic peak between 3200 and 3600 cm −1 attributed to the hydroxyl group due to hydrogen bonding between plant phenolic compound in the EO, carbonyl, and phosphate groups of lecithin. The absorptions at 2977 and 2929, 2900 cm −1 are allocated to symmetric and anti-symmetric vibration of CH 2 in the alkyl chain in EO, tween 20, lecithin, and cholesterol. The spectrum at 1645 cm −1 is attributed to the carbonyl group. The phosphate stretching in 1274 and 1085 cm −1 is attributed to the interaction of compounds in the EO and fatty acid chains or polar heads in the nanoliposome.
ATR-FTIR spectrum of liposome containing A. dracunculus EO (Fig. 3F) displayed the broad band between 3200 to 3600 cm −1 allocated to the OH group due to hydrogen bonding between carboxyl and phosphate groups of lecithin. The absorption at 2976 and 2928 cm −1 are allocated to C-H starching due to SP 3 hybrids of alkane in EO, tween 20, lecithin, and cholesterol. The spectrum at 1644 cm −1 can be attributed to the C=O. The phosphate stretching is presented in 1274 and 1085 cm −1 , and the spectrum at 1044 cm −1 is attributed to C-O.
ATR-FTIR spectrum of liposome containing A. sieberi EO (Fig. 3G) showed the broadband between 3200 to 3600 cm −1 corresponded to hydroxyl groups due to hydrogen bonding interaction between EO, carbonyl, and phosphate groups. Besides, The spectra at 2978 and 2927 cm −1 allocated to C-H starching vibration related to alkanes in EO, Lecithin, tween 20, and cholesterol. Besides, the peak at 1644 cm −1 can be attributed to the C=O. The phosphate stretching presented in 1274 and 1085 cm −1 can be allocated to the interaction of compounds in EO and fatty acid chains or polar heads in nanoliposome. www.nature.com/scientificreports/

Discussions
Excessive use of synthetic pesticides has led to environmental pollution with enhancement in vectors' resistance 38,39 . The development of resistance to insecticides such as pyrethroids, organophosphates, organochlorines, and carbamates has precluded the successful elimination of larval stages 40,41 . For instance, a high rate (78%) of pyrethroids resistance in the WHO African Region has been demonstrated 42 . EOs consist of various natural volatile hydrocarbons and phenylpropenes molecules 43 . Monoterpenes are the main component of EOs which exert neurotoxic effects on insects via AChE and GABA activities 16,44 . However, total EOs confers substantially higher larvicidal or insecticidal effects through multi-target effects 45,46 . So in this study, three EOs from Asteraceae Family was used as larvicides. A. annua L. is a polyphenols-reach plant with antimalarial effects that grows in various geographical and soil pH conditions 47 . Another member of the Asteraceae Family, A.dracunculus has demonstrated larvicidal, antimicrobial, anticancer, and anti-inflammatory effects 48,49 . In addition, Artemisia sieberi has exhibited antimicrobial, antifungal, larvicidal, and insecticidal traits [50][51][52] .
In recent years many reports on using nanostructures containing EOs as mosquito repellents or larvicides have been published. For instance, nanogel containing Zataria multiflora EO with 600 min repellent against An. stephensi compared to 242 min efficacy of DEET 53 57 . The smaller the nanoparticle size, the greater the mobility, and more collisions with larvae (due to Brownian motion) led to better accessibility, permeability, and toxicity against the larval body 58,59 . The current study demonstrated nanoliposomes containing A. dracunculus with LC 50 values of 34 and 23 μg/mL against Ae. aegypti and An. stephensi is a great formulation for use as a mosquito larvicide. Its efficacy is also more than many available reports. For instance, nanoemulsion containing Pterodon emarginatus at 250 μg/mL showed 100% larvicidal effects on Ae. aegypti 60 . Besides, the LC 50 value of Lippia alba nanoemulsion against Ae. aegypti was 31.02 μg/mL 61 . Moreover, the LC 50 value of Myrtus communis nanoemulsion against An. stephensi was reported as 26.1 μg/mL 62 . Besides, Eucalyptus globulus EO nanogel against An. stephensi was reported as 32 μg/mL 63 . The main reason for its considerable capability might be related to the high percentage of estragole (67.6%), which can be used as a larvicidal agent for mosquito control programs. In that way, our findings provide a possible way for further studies to find out the active molecule. However, further investigations must be conducted to describe the mode of action of each constituent independently.

Data availability
The data used to support the findings of this study are included within the article. www.nature.com/scientificreports/